RESUMO
PURPOSE: Dasatinib (BMS-354825) is a potent, oral multi-targeted kinase inhibitor. It is an effective therapy for patients with imatinib-resistant or -intolerant Ph+ leukemias,. It has demonstrated promising preclinical anti-tumor activity, and is under clinical evaluation in solid tumors. To support the clinical development of dasatinib, we identified a pharmacodynamic biomarker to assess in vivo SRC kinase inhibition, with subsequent evaluation in cancer patients. METHODS: The biomarker, phosphorylated SRC (phospho-SRC), was first identified in human prostate PC-3 tumor cells and peripheral blood mononuclear cells (PBMCs) in vitro. It was further assessed in nude mice bearing PC-3 xenografts. Phospho-SRC[pY418] in tumors and PBMC were measured by western blot analysis, and were quantified by ELISA assays. Dasatinib plasma concentrations were determined using LC/MS/MS. RESULTS: In PC-3 cells, dasatinib showed dose-dependent anti-proliferative effect, which correlated with the inhibition of phospho-SRC[pY418] and of SRC kinase activity. With a single oral dose of 50 or 15 mg/kg, tumoral phospho-SRC[pY418] was maximally inhibited at 3 h, partially reversed between 7 and 17 h, and completely recovered after 24 h post dose. At 5 mg/kg, tumoral phospho-SRC[pY418] inhibition was less pronounced and recovered more rapidly to baseline level within 24h. Dasatinib (1 mg/kg) resulted in little inhibition. In PBMCs, a similar time course and extent of phospho-SRC[pY418] inhibition was observed. Inhibition of phospho-SRC[pY418] in vivo appeared to correlate with the preclinical in vivo efficacy and PK profiles of dasatinib in mice. CONCLUSIONS: Phospho-SRC[pY418] may potentially be used as a biomarker to enable assessment of target inhibition in clinical studies exploring dasatinib antitumor activity.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Monitoramento de Medicamentos/métodos , Proteínas de Neoplasias/antagonistas & inibidores , Neoplasias da Próstata/patologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Tiazóis/farmacologia , Quinases da Família src/análise , Adenocarcinoma/química , Adenocarcinoma/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Biomarcadores , Divisão Celular/efeitos dos fármacos , Dasatinibe , Feminino , Humanos , Leucócitos Mononucleares/química , Leucócitos Mononucleares/efeitos dos fármacos , Masculino , Camundongos , Camundongos Nus , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/sangue , Fosfoproteínas/antagonistas & inibidores , Fosforilação , Neoplasias da Próstata/química , Neoplasias da Próstata/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/administração & dosagem , Pirimidinas/farmacocinética , Pirimidinas/uso terapêutico , Organismos Livres de Patógenos Específicos , Especificidade por Substrato , Tiazóis/administração & dosagem , Tiazóis/farmacocinética , Tiazóis/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/sangueRESUMO
PURPOSE: Chronic myeloid leukemia (CML) is caused by reciprocal translocation between chromosomes 9 and 22, forming BCR-ABL, a constitutively activated tyrosine kinase. Imatinib mesylate, a selective inhibitor of BCR-ABL, represents current frontline therapy for CML; however, emerging evidence suggests that drug resistance to imatinib may limit its long-term success. To improve treatment options, dasatinib (BMS-354825) was developed as a novel, oral, multi-targeted kinase inhibitor of BCR-ABL and SRC family kinases. To date, dasatinib has shown promising anti-leukemic activity in preclinical models of CML and in phase I/II clinical studies in patients with imatinib-resistant or imatinib-intolerant disease. EXPERIMENTAL DESIGN: The pharmacokinetic and pharmacodynamic biomarkers of dasatinib were investigated in K562 human CML xenografts grown s.c. in severe combined immunodeficient mice. Tumoral levels of phospho-BCR-ABL/phospho-CrkL were determined by Western blot. RESULTS: Following a single oral administration of dasatinib at a preclinical efficacious dose of 1.25 or 2.5 mg/kg, tumoral phospho-BCR-ABL/phospho-CrkL were maximally inhibited at approximately 3 hours and recovered to basal levels by 24 hours. The time course and extent of the inhibition correlated with the plasma levels of dasatinib in mice. Pharmacokinetic/biomarker modeling predicted that the plasma concentration of dasatinib required to inhibit 90% of phospho-BCR-ABL in vivo was 10.9 ng/mL in mice and 14.6 ng/mL in humans, which is within the range of concentrations achieved in CML patients who responded to dasatinib treatment in the clinic. CONCLUSIONS: Phospho-BCR-ABL/phospho-CrkL are likely to be useful clinical biomarkers for the assessment of BCR-ABL kinase inhibition by dasatinib.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Antineoplásicos/farmacocinética , Biomarcadores Tumorais/antagonistas & inibidores , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas Nucleares/antagonistas & inibidores , Pirimidinas/farmacocinética , Tiazóis/farmacocinética , Proteínas Adaptadoras de Transdução de Sinal/análise , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/sangue , Biomarcadores Tumorais/análise , Western Blotting , Linhagem Celular Tumoral , Dasatinibe , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Proteínas de Fusão bcr-abl/análise , Humanos , Injeções Intravenosas , Camundongos , Camundongos SCID , Proteínas Nucleares/análise , Valor Preditivo dos Testes , Pirimidinas/administração & dosagem , Pirimidinas/sangue , Relação Estrutura-Atividade , Tiazóis/administração & dosagem , Tiazóis/sangue , Fatores de Tempo , Transplante Heterólogo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although Erbitux (cetuximab) has proven therapeutic benefit in the clinical setting, the molecular determinants predicting responsiveness to this agent are still not very well understood. Here, we assessed the relationship between basal total and activated (pY1068) epidermal growth factor receptor (EGFR) levels in a tumor and the responsiveness to cetuximab monotherapy or combination-based treatment using human xenograft models. Cetuximab treatment alone (0.25-1 mg/mouse/injection, q3d, i.p.) effectively delayed the growth of GEO and L2987 tumors by a minimum of 10 days corresponding to log cell kill values of >or=1.0. Borderline activity was seen in the A549 and WiDr xenografts. However, cetuximab failed to show any significant antitumor activity in the HT29, HCT116, LOVO, Colo205, LX-1, HCC70, and N87 models. All of the studied tumors had detectable yet variable levels of EGFR. For combination regimens, cetuximab (1 mg/mouse/injection, q3dx5, i.p.) and cisplatin (4.5 mg/kg/injection, q3dx5, i.v.) proved to be significantly more efficacious than individual monotherapies in the cisplatin-refractory yet cetuximab-responsive GEO tumor model (P < 0.001). However, no therapeutic enhancement was observed in the cisplatin and cetuximab weakly responsive A549 xenograft. Similarly, combinations of CPT-11 (48 mg/kg/injection, q3dx5, i.v.) with cetuximab (1 mg/mouse/injection, q3dx5, i.p.) failed to show any improvements over individual monotherapies in the cetuximab resistant/weakly responsive HT29, A549, and WiDr models. We conclude that preclinical activity associated with cetuximab monotherapy does not correlate directly with relative basal levels of total or activated (pY1068) EGFR in a tumor. Moreover, robust single-agent activity by cetuximab may be the best predictor for this agent to potentiate chemotherapy-mediated antitumor activities.
Assuntos
Anticorpos Monoclonais/farmacologia , Receptores ErbB/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Antineoplásicos , Protocolos de Quimioterapia Combinada Antineoplásica , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Linhagem Celular Tumoral , Cetuximab , Cisplatino/administração & dosagem , Receptores ErbB/efeitos dos fármacos , Feminino , Humanos , Irinotecano , Camundongos , Camundongos Nus , Neoplasias Experimentais/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Epidermal growth factor receptor (EGFR), a protein tyrosine kinase expressed in many types of human cancers, has been strongly associated with tumor progression. Cetuximab is an IgG(1) anti-EGFR chimeric mouse/human monoclonal antibody that has been approved for the treatment of advanced colon cancer. Using human tumor xenografts grown in nude mice, we have determined the in vivo pharmacodynamic response of cetuximab at efficacious doses. Three pharmacodynamic end points were evaluated: tumoral phospho-EGFR, tumoral mitogen-activated protein kinase (MAPK) phosphorylation, and Ki67 expression. EXPERIMENTAL DESIGN: The pharmacodynamic study was conducted in nude mice bearing Geo tumors following a single i.p. administration of 0.25 and 0.04 mg. The tumors were analyzed by immunohistochemistry. The levels of phospho-EGFR were quantitated by an ELISA assay. RESULTS: At 0.25 mg, phospho-EGFR was maximally inhibited by 91% at 24 hours, whereas the level of inhibition decreased to 72% by 72 hours. At 0.04 mg, the maximum inhibition of phospho-EGFR was 53% at 24 hours, whereas the level of inhibition decreased to 37% by 72 hours. The time course of phospho-EGFR inhibition and recovery seemed to correlate with the pharmacokinetics of cetuximab. Immunohistochemical analysis showed that phospho-MAPK and Ki67 expression were inhibited between 24 and 72 hours at 0.25 and 0.04 mg. A pharmacokinetic/pharmacodynamic model was established and predicted that the plasma concentration of cetuximab required to inhibit 90% of phospho-EGFR was 67.5 mug/mL. CONCLUSIONS: Phospho-EGFR/phospho-MAPK could be useful clinical biomarkers to assess EGFR inhibition by cetuximab.
Assuntos
Antineoplásicos/farmacocinética , Biomarcadores Tumorais , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Cetuximab , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/metabolismo , Feminino , Humanos , Imunoglobulina G/química , Imuno-Histoquímica , Antígeno Ki-67/biossíntese , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Nus , Transplante de Neoplasias , Fosforilação , Fatores de TempoRESUMO
The insulin-like growth factor I receptor (IGF-IR) is a transmembrane tyrosine kinase that is essential to growth and development and also thought to provide a survival signal for the maintenance of the transformed phenotype. There has been increasing interest in further understanding the role of IGF-I signaling in cancer and in developing receptor antagonists for therapeutic application. We describe herein a novel animal model that involves transgenic expression of a fusion receptor that is constitutively activated by homodimerization. Transgenic mice that expressed the activated receptor showed aberrant development of the mammary glands and developed salivary and mammary adenocarcinomas as early as 8 weeks of age. Xenograft tumors and a cell line were derived from the transgenic animals and are sensitive to inhibition by a novel small-molecule inhibitor of the IGF-IR kinase. This new model should provide new opportunities for further understanding how aberrant IGF-IR signaling leads to tumorigenesis and for optimizing novel antagonists of the receptor kinase.
Assuntos
Adenocarcinoma/genética , Transformação Celular Neoplásica/genética , Neoplasias Mamárias Experimentais/genética , Receptor IGF Tipo 1/biossíntese , Neoplasias das Glândulas Salivares/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Antígenos CD8/biossíntese , Antígenos CD8/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Feminino , Humanos , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Nus , Camundongos Transgênicos , Transplante de Neoplasias , Gravidez , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Neoplasias das Glândulas Salivares/enzimologia , Neoplasias das Glândulas Salivares/patologia , Transfecção , Transgenes/genética , Transplante HeterólogoRESUMO
Tetrahydroquinoline-based small molecule inhibitors of farnesyltransferase (FT) have been identified. Lead compounds were shown to have nanomolar to sub-nanomolar activity in biochemical assays with excellent potency in a Ras-mutated cellular reversion assay. BMS-316810 (9e), a 0.7 nM FT inhibitor, was orally-active in a nude mouse tumor allograft efficacy study.